Project Summary Genome editing technologies, such CRISPR/Cas9 provide a rapid, and targeted means of both knocking out gene expression and knocking in gene modifications. However, the current workflow required for CRISPR cell line generation relies on several technologies which reduce throughput, efficiency and the overall viability of genome edited cells. Cell Microsystems has developed a single cell isolation and recovery platform ideally suited to high-throughput production of CRISPR cell lines. The core technology comprises a disposable microwell array (the CellRaft? Array) on which cells are seeded and imaged. To isolate single cells, a motorized needle penetrates the resealable elastomeric underside of the Array to displace the individual CellRaft from its microwell. The CellRaft material is loaded with magnetic nanoparticles, allowing retrieval of the CellRaft, and attached cell, using a magnetic wand. By enabling on-array transfection and recovery, the system replaces harmful trypsinization, re-plating/recovery steps and stressful sheer forces used in fluorescence-assisted cell sorting (FACS), resulting in a method amenable to rapid high-throughput CRISPR cell line generation. During Phase I we plan to build on work by one of our Early Adopter Program participants and Principal Investigator of this program, William Marzluff, Ph.D. of the University of North Carolina at Chapel Hill. Using the CellRaft Array, Dr. Marzluff?s team has streamlined the transfection and cloning workflow for producing CRISPR cell lines. Thus far, his team has: 1) developed a clonal colony isolation protocol using the CellRaft System; 2) shown comparable viability of clonal colonies isolated on the CellRaft system compared to FACS and 3) established a protocol for transfecting cells pre-seeded on the CellRaft Array, eliminating re-plating and recovery steps required for FACS. Here we will expand on this work by 1) optimizing transfection and sorting on the array for CRISPR/Cas9 genome editing and 2) developing a 24-well CellRaft Array allowing a dramatic increase in sorting throughput over other clonal isolation technologies including FACS. Pending successful development of these methods in Phase I, our Phase II program will focus on implementing the workflow on our under development automated instrument, the Automated Isolation and Retrieval, or AIR? System. Automating the workflow will allow higher throughput isolation of clonal colonies from multiple transfection reactions (384 total clones in 2 hours), as well as semi-quantitative sorting of cells using fluorescent marker intensities. Also during Phase II we will carry out a CRIPSR/Cas9 screen using hundreds of sgRNAs on the AIR? System. CRISPR/Cas9 screening is likely to be one of the primary market drivers of this technology, and using the powerful imaging capabilities of the AIR? System will allow screening for more sophisticated phenotypes than merely cell death.